The ability of DNA to form triple helices between duplex DNA and single strands of appropriate sequence offers promise as a developing technique for sequence-specific DNA cleavage and the modulation of gene expression, including therapeutic uses. The major limitation of this approach has been that triplex formation only occurs at sequences that are largely or entirely homopurine tracts. The proposed research will investigate new approaches for the loosening of this requirement, with the ultimate goal being the capability to achieve triplex formation at any sequence. One new approach involves the pairing of a single strand with both strands of a mixed sequence duplex, with Py.PuPy base triplets being formed when purines are on one strand of the Watson-Crick duplex and Pu.PuPy triplets when that are on the other strand. This approach maintains the required polarity for both pairing motifs without requiring any unusual linkages or base analogs. Preliminary studies on intramolecular triplex formation have demonstrated the promise of this strategy for PuPuPuPuPyPyPyPy sequences. The proposed research will examine to what extent this strand-switching approach can obviate the need for homopurine sequences. A pool of single-stranded DNA molecules of random sequence (up to 1010- 1012 different sequences) will also be screened to select the best sequences for triplex formation with mixed sequence targets of various types. Triplex formation will be employed to control gene expression at the DNA and RNA level in the human immunodeficiency virus (HIV-1). A novel approach based on triplex formation (triplex-mediated antisense) is proposed for arresting gene expression at the RNA level.